Cyanobactins are ribosomally produced and post-translationally modified peptides (RiPPs). Their functions are not known, but some are likely to be important in symbiotic relationships of producing cyanobacteria with host animals, and some have micromolar toxicity. In nature, highly conserved cyanobactin pathways produce many different products, and each pathway studied in the lab so far has the ability to process widely varying peptides. The goal of this research is to advance understanding of the substrate selectivity of the final modifying enzyme in many cyanobactin pathways, and to advance bioengineering efforts aimed at making new cyanobactins. The vast product diversity available from each pathway and the known activities of some cyanobactin natural products makes cyanobactin pathways an attractive system for producing new drug leads. Cyanobactin biosynthetic pathways consist of precursor peptides and modifying enzymes. The precursor peptides contain enzyme recognition sequences and core sequences that become the final products. The modifying enzymes carry out post-translational tailoring reactions such as proteolysis, cyclization of individual amino acids and of the whole peptide, oxidations, methylation and prenylation. Underlying the wide product variability is the ability of every enzyme in a pathway to accept substrates with widely differing amino acid sequences in their cores. My specific aims are 1) to test the role of enzyme and substrate residues in substrate selectivity of cyanobactin prenyltransferases, 2) to study the mechanism(s) through which particular growth conditions increase heterologous cyanobactin production in E. coli and 3) to express new cyanobactins in E. coli, including variants on cyanobactins known to be active, for use in structure- activity relationship studies. The experimental approach involves biochemical work on purified prenyltransferases, supported by x-ray crystallography studies being done in another lab, engineering pathways for expression in E. coli, testing various expression conditions, and purifying new cyanobactin products.